Protein-DNA selectivity is a central event in many biological processes. Type II restriction enzymes are ideal systems for studying selectivity, due to their high specificity and striking variety. The enzymes recognize and cleave DNA sequences that vary between four to eight base pairs. Their specificity is remarkable. A single base pair change within the recognition sequence can lead to well over a million fold reduction in activity. An understanding of sequence specific cleavage is relevant to proteins mediating site-specific recombination and DNA repair by excision. The long term goals of this project are to understand the mechanisms by which type II restriction enzymes recognize and cleave DNA, and the design of mutants with altered DNA specificities. To accomplish these goals the specific aims of this proposal are: 1) To determine by X-ray crystallography, structures that mimic the various stages of the BamHI reaction pathway. These include the double-cleaved intermediate, the single-cleaved intermediate, and non-specific complexes. The double-cleaved intermediate will be prepared by soaking the native cocrystals in MnCl2 solutions. The single-cleaved intermediate will be prepared by hybridizing a non-cleavable DNA strand with a cleavable strand. Non-specific complexes will be prepared by crystallizing BamHI with DNA fragments, that contain sequences other than the BamHI recognition sequence GGATCC. These structures will provide a complete, dynamic view of the sequence of events underlying site- specific deavage. The known structures of BamHI and BamHI/specific DNA complex will assist in the structure determination. 2) To determine the structure of FokI-DNA complex. FokI is an unusual restriction enzyme in that the DNA recognition and cleavage functions are located on distinct domains. X-ray data extending to 2.8 Angstroms resolution have been measured from a native cocrystal, and a mercurial derivative identified. Crystals of the enzyme by itself have also been obtained. 3) To create artificial restriction enzymes, by attaching the cleavage domain of FokI to the DNA binding domains of transcription factors. These hybrid enzymes will be particularly useful in the mapping of large genomes. 4) To determine the structure of SfiI-DNA complex. SfiI is one of the few restriction enzymes that can recognize eight DNA base-pairs, and its mode of action may resemble that of proteins mediating site-specific recombination. The enzyme has already been cocrystallized with DNA, and efforts to prepare heavy atom derivatives are well underway.